Compounds and methods of use thereof for treating tumors

ABSTRACT

The invention encompasses novel compounds and pharmaceutically acceptable salts thereof and compositions including therapeutically or prophylactically effective amounts of such compounds or pharmaceutically acceptable salts thereof. The invention also encompasses methods for treating or preventing diseases and disorders associated with abnormal cell growth, for example, treating or preventing cancer or tumor growth, which methods include administering to a mammal in need thereof a composition comprising a therapeutically or prophylactically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Part of the work performed during development of this invention utilizedU.S. Government funds under National Institutes of Health (NationalCancer Institute) Grant Nos. R01-CA82599 and R01-CA150646. The U.S.Government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

N/A

BACKGROUND OF THE INVENTION

Field of the Invention

The invention encompasses novel compounds and pharmaceuticallyacceptable salts thereof and compositions including therapeutically orprophylactically effective amounts of such compounds or pharmaceuticallyacceptable salts thereof.

Background of the Invention

The BRCA1 gene was identified in 1994 based its linkage to hereditarybreast and ovarian cancers. BRCA1 mutations also confer an increasedrisk for several other hormone-responsive tumor types (cervical,endometrial, and prostate cancers). BRCA1 is a protein with 1863 aminoacids and is considered to be a nuclear phospho-protein with conservedN-terminal RING and C-terminal acidic transcriptional activationdomains. A BRCA1 null mutation confers early embryonic lethality (day7.5-8.5) in mice, due to a severe proliferation defect caused by p53activation and consequent p21WAF1 expression, but a partial BRCA1deletion targeted to the mammary gland allows survival with developmentof breast cancer. In mice, BRCA1 expression is widespread, but it isespecially increased in rapidly proliferating cells in compartments thatare also undergoing differentiation, including mammary epithelial cellsduring puberty and pregnancy.

Various functional activities are ascribed to BRCA1, but it is stillunclear which of these are essential for cancer suppression. Earlystudies indicated that BRCA1 is expressed and phosphorylated cyclically,suggesting a role in cell cycle regulation. BRCA1 was found to interactwith RB1 and to collaborate with RB1 in regulating progression from G1to S phase. Most studies on BRCA1 function, however, have concentratedon its role in DNA repair. In response to DNA damage, BRCA1 isphosphorylated by several up-stream protein kinases (ATM(ataxia-telangiectasia mutated), ATR, and CHEK2) and associates with theRMN complex (Rad50-Mre11-p95NBS1) in radiation-induced nuclear “dots,”suggesting a role in the repair of double-strand breaks. BRCA1deficiency confers defects in several types of DNA repair mechanisms,including microhomology-dependent repair and homologous recombination.Studies of BRCA1 mutant cell types have also established roles for BRCA1in several DNA damage-responsive cell cycle checkpoints (intra-S andG2/M). BRCA1 mutant cells exhibit evidence of genomic instability, e.g.,centrosome amplification, aneuploidy, and chromosomal aberrations. Inaddition, BRCA1 is present in a large multi-protein BRCA1-associatedgenome surveillance complex (BASC). For these reasons, BRCA1 is thoughtto mediate a “caretaker” function in the maintenance of genomicstability.

Estrogen receptor-alpha (ER-α) is a member of the nuclear receptorsuperfamily of ligand activated transcription factors, characterized by:an N-terminal transactivation domain (AF-1), a conserved C-terminalactivation domain (AF-2), which overlaps with the ligand binding domain(LBD), a sequence-specific DNA-binding domain (DBD), and a hinge regionlocated between the DBD and AF-2 regions. BRCA1 is a strong inhibitor ofE2-stimulated ER-α activity via a direct physical interaction with theAF-2 activation domain of ER-α. BRCA1 also represses ligand-independentactivation of ER-α, since BRCA1-siRNA can stimulate ER-α activity in theabsence of estrogen. This finding suggests that the endogenous levels ofBRCA1 are sufficient to inhibit basal activity levels of ER-α. Furtherstudies have documented that BRCA1 broadly inhibits E2-stimulated geneexpression and blocks E2-stimulated proliferation of ER-α positive humanbreast cancer cells. BRCA1 has been detected at the ERE site ofestrogen-regulated promoters (pS2 and cathepsin D), and exposure to E2causes a rapid loss of BRCA1 from this site. Various breast cancer-associated BRCA1 mutations abrogate or greatly lessen the ability ofBRCA1 to inhibit ER-α, suggesting that this function is essential forbreast cancer suppression. Finally, it has recently been shown BRCA1 caninhibit the activity of aromatase (CYP19A1), a cytochrome P450 enzymethat mediates the conversion of androgens into estrogens, in epithelialcells and adipocytes.

About two-thirds of human sporadic (non-hereditary) breast cancers areER-α positive, and hormonal factors clearly affect the risk fordeveloping these tumors. In contrast to sporadic cancers, abouttwo-thirds of BRCA1 mutant human breast cancers are ER-α negative.However, there is substantial evidence from clinical/epidemiologicstudies and from experimental animal studies suggesting a hormonaletiology for BRCA1-mutant breast cancers, even though the tumors usuallyend up being ER-α negative.

For example, several studies provide evidence that BRCA1-mutant mammarycarcinogenesis is estrogen-sensitive. For example, the chemopreventionagent Tamoxifen, which is a partial ER-α antagonist and agonist, cancause an increase in the prevalence of mammary hyperplasia andaccelerated the development of mammary cancer inBRCA1^(Co/Co)/MMTV-Cre/p53^(+/−) mice. These findings were consistentwith the finding that in MCF-7 cells, BRCA1 knockdown shifted thebalance of Tamoxifen activity from ER-α antagonist to agonist. Moreover,in BRCA1^(Co/Co)/MMTV-Cre/p53^(+/−) mice, bilateral ovariectomysignificantly reduced the incidence of mammary cancer. The ovariectomywas most effective in reducing cancer risk when performed well beforethe time the tumors normally occur. These results suggest that the earlystages of BRCA1-dependent mammary tumorigenesis could be E2-dependent,and thus it may be possible to prevent or treat BRCA1-mutant breastcancers using an agent that can mimic the ability of BRCA1 to inhibitER-α activity.

SUMMARY OF THE INVENTION

The invention relates to compounds, compositions and methods of usethereof of compounds of Formulas I-III.

In one embodiment, the invention is directed towards compounds ofFormula I:

or pharmaceutically acceptable salts or prodrugs thereof, wherein

-   R₁, R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H; a halogen;    —OH; an amine; a carboxyl; substituted or unsubstituted: cyclic or    acyclic alkyl group, cyclic or acyclic alkenyl group, cyclic or    acyclic akynyl group, carboxyalkyl, cyclic or acyclic alkoxy, aryl    group, alkylaryl group, arylalkyl group, heteroaryl group,    heteroalkyl group; COR₁₀; CSR₁₀; SOR₁₀; SO₂R₁₀; CON(R₁₀)₂;    CSN(R₁₀)₂; wherein R₅ and R₆, or R₃ and R₄, or R₇ and R₈ can form a    3-10 membered substituted or unsubstituted ring with one or more of    C, S, O, or N,-   R₂ is CH₂, CHR₁, COR₁, CSR₁, SO, SO₂, SHOR₁, NH, NOR₁ or O,-   R₉ is CO, O, NH or CH₂, and-   R₁₀ is H; —OH; an amine; a carboxyl; substituted or unsubstituted:    cyclic or acyclic alkyl group, cyclic or acyclic alkenyl group,    cyclic or acyclic akynyl group, an aryl group, an alkylaryl group,    an arylalkyl group, heteroaryl group, heteroalkyl group; or a    halogen.

In another embodiment, the invention is directed towards compounds ofFormula II:

or pharmaceutically acceptable salts or prodrugs thereof, wherein

-   R₁ is H; a halogen; —OH; an amine; a carboxyl; substituted or    unsubstituted: cyclic or acyclic alkyl group, cyclic or acyclic    alkenyl group, cyclic or acyclic akynyl group, carboxyalkyl, cyclic    or acyclic alkoxy, aryl group, alkylaryl group, arylalkyl group,    heteroaryl group, heteroalkyl group; COR₈; CSR₈; SOR₈; SO₂R₈;    CON(R₈)₂; CSN(R₈)₂; wherein R₁ and R₄, can form a 3-10 membered ring    with one or more of C, S, O or N;-   R₂ and R₅ are each independently H; a halogen; —OH; an amine; a    carboxyl; substituted or unsubstituted: cyclic or acyclic alkyl    group, cyclic or acyclic alkenyl group, cyclic or acyclic alkynyl    group, carboxyalkyl, cyclic or acyclic alkoxy, aryl group, alkylaryl    group, arylalkyl group, heteroaryl group, heteroalkyl group; COR₈;    CSR₈; SOR₈; SO₂R₈; CON(R₈)₂; CSN(R₈)₂; or a monocyclic or polycyclic    substituted or unsubstituted fused ring structure that is fused with    the ring atoms to which either R₂ or R₅ are attached; wherein R₂ and    R₅ can form a 3-10 membered ring with one or more of C, S, O or N;-   R₃ and R₄ are each independently CH; C(R₁)₂; CO; COR₁; CON(R₁)₂; CS;    CSR₁; CS(R₁)₂; C(SR₁)₁; CSNR₁; SH; SO; SO₂; S(O)R₁; S(O)(R₁)₂; N;    NH; NR₁; NOR₁; or O; wherein R₃ and R₄, together with the ring atoms    to which R₄ is attached, can form an additional fused 5- or    6-membered ring structure, with one or more of C, S, O or N; wherein    R₂ and R₃ together with the ring atoms to which they are attached,    can form an additional fused 5- or 6-membered ring structure, with    one or more of C, S, O or N; wherein R₃ together with the ring atoms    to which it is attached, can form an additional fused 5- or    6-membered ring structure, with one or more of C, S, O or N;-   R₈ is H; —OH; an amine; a carboxyl; substituted or unsubstituted:    cyclic or acyclic alkyl group, cyclic or acyclic alkenyl group,    cyclic or acyclic akynyl group, an aryl group, an alkylaryl group,    an arylalkyl group, heteroaryl group, heteroalkyl group; or a    halogen; and-   is a single bond or a double bond, wherein if    is a double bond, R₃ and R₄ are independently CH, N or SH.

In another embodiment, the invention is directed towards compounds ofFormula III:

or pharmaceutically acceptable salts or prodrugs thereof, wherein

-   R₁ and R₃ are each independently H; a halogen; —OH; an amine; a    carboxyl; substituted or unsubstituted: cyclic or acyclic alkyl    group, cyclic or acyclic alkenyl group, cyclic or acyclic akynyl    group, carboxyalkyl, cyclic or acyclic alkoxy, aryl group, alkylaryl    group, arylalkyl group, heteroaryl group, heteroalkyl group; COR₈;    CSR₈; SOR₈; SO₂R₈; CON(R₈)₂; CSN(R₈)₂; wherein R₁ and R₃ can form a    6-15 membered ring with one or more of C, S, O, or N,-   R₂ and R₄ are each independently H; a halogen; —OH; an amine; a    carboxyl; substituted or unsubstituted: cyclic or acyclic alkyl    group, cyclic or acyclic alkenyl group, cyclic or acyclic akynyl    group, carboxyalkyl, cyclic or acyclic alkoxy, aryl group, alkylaryl    group, arylalkyl group, heteroaryl group, heteroalkyl group; COR₈;    CSR₈; SOR₈; SO₂R₈; CON(R₈)₂; CSN(R₈)₂; wherein R₂ and R₄ can form a    monocyclic or polycyclic 3-14 membered fused ring structure,    together with the atoms to which they are attached, with one or more    of C, S, O, or N, and-   R₈ is —H; —OH; an amine; a carboxyl; substituted or unsubstituted:    cyclic or acyclic alkyl group, cyclic or acyclic alkenyl group,    cyclic or acyclic akynyl group, an aryl group, an alkylaryl group,    an arylalkyl group, heteroaryl group, heteroalkyl group; or a    halogen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts cellular activity of select compounds of the presentinvention with a luciferase reporter assay from an estrogen responseelement (ERE) in the MCF-7 human breast cancer cell line.

FIG. 2 depicts cellular activity of select compounds of the presentinvention with a luciferase reporter assay from an estrogen responseelement (ERE) in the MCF-7 human breast cancer cell line.

DETAILED DESCRIPTION OF THE INVENTION

As used herein and unless otherwise indicated, the phrase “compound(s)of the invention” means, collectively, any of the compounds of FormulasI, II and III and pharmaceutically acceptable salts thereof. Examples ofcompounds of the present invention include but are not limited to thecompounds depicted herein. The compounds of the invention are identifiedherein by their chemical structure and/or chemical name. Where acompound is referred to by both a chemical structure and a chemicalname, and that chemical structure and chemical name conflict, thechemical structure is determinative of the compound's identity. Thecompounds of the invention may contain one or more chiral centers and/ordouble bonds and, therefore, exist as stereoisomers, such as double-bondisomers, i.e., geometric isomers, enantiomers, or diastereomers.According to the invention, the chemical structures depicted herein, andtherefore the compounds of the invention, encompass all of thecorresponding compounds' enantiomers and stereoisomers, i.e., both thestereomerically pure forms and enantiomeric and stereoisomeric mixtures.The term “stereomerically pure” is understood in the art and is used tomean geometrically pure, enantiomerically pure, or diastereomericallypure compounds. Enantiomeric and stereoisomeric mixtures can be resolvedinto their component enantiomers or stereoisomers by well known methods,such as chiral-phase gas chromatography, chiral-phase high performanceliquid chromatography, crystallizing the compound as a chiral saltcomplex, or crystallizing the compound in a chiral solvent. Enantiomersand stereoisomers can also be obtained from stereomerically- orenantiomerically-pure intermediates, reagents, and catalysts by wellknown asymmetric synthetic methods.

As used herein and unless otherwise indicated, the term “alkoxy group”means an —O-alkyl group, wherein alkyl is as defined herein. The alkoxygroup can be cyclic or acyclic and can be unsubstituted or substitutedwith one or two suitable substituents. In one embodiment, the alkylchain of an alkyloxy group is from 1 to 6 carbon atoms in length,referred to herein, for example, as “(C₁-C₁₀)alkoxy.”

As used herein, the term “substituted” and “a suitable substituent” whenused in conjunction with a substituent of the compounds of the presentinvention, refers to a substituent, e.g., an alkyl or aryl, in which oneor more hydrogen atom bound to any carbon or heteroatom is replaced byanother group. The “substitutes” and the “suitable substituents” aregroups that do not nullify the synthetic or pharmaceutical utility ofthe compounds of the invention or the intermediates useful for preparingthem. Examples of substituted groups or suitable substituents include,but are not limited to: (C₁-C₈)alkyl; (C₁-C₈)alkenyl; (C₁-C₈)alkynyl;(C₆)aryl; (C₃-C₅)heteroaryl; (C₃-C₇)cycloalkyl; (C₁-C₈)alkoxy;(C₆)aryloxy; —CN; —OH; SH, oxo; halo, —NO₂, —CO₂H; —NH₂; —NHOH,—NH((C₁-C₈)alkyl); —N((C₁-C₈)alkyl)₂; —NH((C₆)aryl); —NHO((C₁-C₈)alkyl);—N(O(C₁-C₈)alkyl)₂; —NH(O(C₆)aryl); —S((C₁-C₈)alkyl); —S((C₁-C₈)alkyl)₂; —S((C₆)aryl); (═O); C(S), —N((C₆)_(aryl))₂; —CHO;—C(O)((C₁-C₈)alkyl); —C(O)((C₅)aryl); —CO₂((C₁-C₈)alkyl); and—CO₂((C₆)aryl), —C(S)((C₁-C₈)alkyl); —C(S)((C₆)aryl);—SO₂((C₁-C₈)alkyl); —SO₂((C₆)aryl), and —SO₃H, —C(S)O((C₁-C₈)alkyl);—C(S)(O)((C₆)aryl). In additional illustrative embodiments, thesubstituents can be one or more than one suitable groups, such as, butnot limited to, —F, —Cl, —Br, —I, —OH, azido, —SH, alkyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl,substituted heterocycloalkyl, heteroalky, alkyoxyl, alkylhalos, e.g.,CF₃, alkylthiol, amino, phosphido, hydroxylamino, N-alkylamino,—N,N-dialkylamino, —N,N-dimethylamino, acyl, alkyloxycarbonyl, sulfonyl,urea, —NO₂, triazolyl, thio, and combinations thereof. One of skill inart can readily choose a suitable substituent based on the stability andpharmacological and synthetic activity of the compound of the invention.

As used herein and unless otherwise indicated, the term “alkenyl group”means a monovalent unbranched or branched hydrocarbon chain having oneor more double bonds therein. The double bond of an alkenyl group can beunconjugated or conjugated to another unsaturated group. Suitablealkenyl groups include, but are not limited to (C₂-C₈)alkenyl groups,such as vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl,pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl,4-(2-methyl-3-butene)-pentenyl. The alkenyl group can be cyclic oracyclic and can be unsubstituted or substituted with one or two suitablesubstituents.

As used herein and unless otherwise indicated, the term “heteroalkenyl”is an alkenyl group as defined herein having at least one heteroatomincorporated within the alkenyl group. In certain embodiments, one ofthe hydrogen atoms can be substituted with a group having a heteroatom.For example, the group can be a hydroxyl group (OH) or thiol group (SH).Other examples of groups containing one or more heteroatoms include butare not limited to, nitro, amino, ester, carboxylic acid, carbamide,sulfonate, sulfonic acid, alkoxy, or SO₂R or S(O)₂OR, where R can behydrogen or an alkyl group described above. Alternatively, one of thecarbon atoms of the alkyl group can be substituted with a heteroatom.Examples of heteroatoms include, but are not limited to, nitrogen,oxygen, sulfur, and phosphorous. The heteroalkenyl group can be cyclicor acyclic and can be substituted or unsubstituted.

As used herein and unless otherwise indicated, the term “alkylalkoxy” or“alkyloxyalkyl group” means a saturated, monovalent unbranched orbranched hydrocarbon chain covalently bonded to an oxygen and covalentlybonded to a second a saturated, monovalent unbranched or branchedhydrocarbon chain (e.g., -alkyl-O-alkyl). The alkyloxy group can becyclic or acyclic and can be substituted or unsubstituted.

As used herein and unless otherwise indicated, the term “alkyl” or“alkyl group” means a substituted or unsubstituted, saturated,monovalent unbranched or branched hydrocarbon chain. Examples of alkylgroups include, but are not limited to, (C₁-C₁₀)alkyl groups, such asmethyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl,2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl,2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl,4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl,2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,and hexyl, and longer alkyl groups, such as heptyl, and octyl. An alkylgroup can be unsubstituted or substituted with one or two suitablesubstituents.

As used herein and unless otherwise indicated, the term “heteroalkyl” isan alkyl group as defined herein having at least one heteroatomincorporated within the alkyl group. In certain embodiments, one of thehydrogen atoms can be substituted with a group having a heteroatom. Forexample, the group can be a hydroxyl group (OH) or thiol group (SH).Other examples of groups containing one or more heteroatoms include butare not limited to, nitro, amino, ester, carboxylic acid, carbamide,sulfonate, sulfonic acid, alkoxy, or SO₂R or S(O)₂OR, where R can behydrogen or an alkyl group described above. Alternatively, one of thecarbon atoms of the alkyl group can be substituted with a heteroatom.Examples of heteroatoms include, but are not limited to, nitrogen,oxygen, sulfur, and phosphorous. The heteroalkyl group can be cyclic oracyclic and can be substituted or unsubstituted.

As used herein and unless otherwise indicated, the term “alkynyl group”means monovalent unbranched or branched hydrocarbon chain having one ormore triple bonds therein. The triple bond of an alkynyl group can beunconjugated or conjugated to another unsaturated group. Suitablealkynyl groups include, but are not limited to, (C₂-C₆)alkynyl groups,such as ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl,4-methyl-1-butynyl, 4-propyl-2-pentynyl, and 4-butyl-2-hexynyl. Analkynyl group can be unsubstituted or substituted with one or twosuitable substituents.

As used herein and unless otherwise indicated, the term “heteroalkynyl”is an alkynyl group as defined herein having at least one heteroatomincorporated within the alkynyl group. In certain embodiments, one ofthe hydrogen atoms can be substituted with a group having a heteroatom.For example, the group can be a hydroxyl group (OH) or thiol group (SH).Other examples of groups containing one or more heteroatoms include butare not limited to, nitro, amino, ester, carboxylic acid, carbamide,sulfonate, sulfonic acid, alkoxy, or SO₂R or S(O)₂OR, where R can behydrogen or an alkyl group described above. Alternatively, one of thecarbon atoms of the alkyl group can be substituted with a heteroatom.Examples of heteroatoms include, but are not limited to, nitrogen,oxygen, sulfur, and phosphorous. The heteroalkynyl group can be cyclicor acyclic and can be substituted or unsubstituted.

As used herein and unless otherwise indicated, the term “amine” caninclude a primary, a secondary and/or a tertiary amine group.

As used herein and unless otherwise indicated, the term “aryl group”means a monocyclic or polycyclic-aromatic radical comprising carbon andhydrogen atoms. Examples of suitable aryl groups include, but are notlimited to, phenyl, tolyl, anthacenyl, fluorenyl, indenyl, azulenyl, andnaphthyl, as well as benzo-fused carbocyclic moieties such as5,6,7,8-tetrahydronaphthyl. An aryl group can be unsubstituted orsubstituted with one or two suitable substituents. In one embodiment,the aryl group is a monocyclic ring, wherein the ring comprises 6 carbonatoms, referred to herein as “(C₆)aryl.”

As used herein and unless otherwise indicated, the term “phenyl” means—C₆H₅. A phenyl group can be unsubstituted or substituted with one ortwo suitable substituents.

As used herein and unless otherwise indicated, the term “heteroarylgroup” means a monocyclic- or polycyclic aromatic ring comprising carbonatoms, hydrogen atoms, and one or more heteroatoms, for example 1 to 3heteroatoms, independently selected from nitrogen, oxygen, and sulfur.Illustrative examples of heteroaryl groups include, but are not limitedto, pyridinyl, pyridazinyl, pyrimidyl, pyrazyl, triazinyl, pyrrolyl,pyrazolyl, imidazolyl, (1,2,3,)- and (1,2,4)-triazolyl, pyrazinyl,pyrimidinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, furyl,phienyl, isoxazolyl, and oxazolyl. A heteroaryl group can beunsubstituted or substituted with one or two suitable substituents. Inone embodiment, a heteroaryl group is a monocyclic ring, wherein thering comprises 2 to 5 carbon atoms and 1 to 3 heteroatoms, referred toherein as “(C₂-C₅)heteroaryl.”

As used herein, the term “benzyl” means —CH₂-phenyl.

As used herein and unless otherwise indicated, the term “aryloxy group”means an —O-aryl group, wherein aryl is as defined herein. An aryloxygroup can be unsubstituted or substituted with one or two suitablesubstituents. In one embodiment, the aryl ring of an aryloxy group is amonocyclic ring, wherein the ring comprises 6 carbon atoms, referred toherein as “(C₆)aryloxy.”

As used herein, the term “carbonyl” group is a divalent group of theformula —C(O)—.

As used herein and unless otherwise indicated, the term “carboxyalkyl”means an alkyl group as defined herein that has been substituted with atleast one carboxy group.

As used herein and unless otherwise indicated, the terms “cyclic alkyl”and “cycloalkyl group” are used interchangeably and mean a monocyclic orpolycyclic saturated ring comprising carbon and hydrogen atoms andhaving no carbon-carbon multiple bonds. Examples of cycloalkyl groupsinclude, but are not limited to, (C₃-C₇)cycloalkyl groups, such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, andsaturated cyclic and bicyclic terpenes. A cycloalkyl group can beunsubstituted or substituted by one or two suitable substituents. In oneembodiment, the cycloalkyl group is a monocyclic ring or bicyclic ring.

As used herein and unless otherwise indicated, the term“heterocycloalkyl group” means a monocyclic or polycyclic ringcomprising carbon and hydrogen atoms and at least one heteroatom, forexample, 1 to 3 heteroatoms selected from nitrogen, oxygen, and sulfur,and having no unsaturation. Examples of heterocycloalkyl groups includebut are not limited to pyrrolidinyl, pyrrolidino, piperidinyl,piperidino, piperazinyl, piperazino, morpholinyl, morpholino,thiomorpholinyl, thiomorpholino, and pyranyl. A heterocycloalkyl groupcan be unsubstituted or substituted with one or two suitablesubstituents. In one embodiment, the heterocycloalkyl group is amonocyclic or bicyclic ring. In a more specific embodiment, theheterocycloalkyl group is a monocyclic ring, wherein the ring comprisesfrom 3 to 7 carbon atoms and from 1 to 3 heteroatoms, referred to hereinas “(C₁-C₇)heterocycloalkyl.”

As used herein and unless otherwise indicated, the terms “cyclicalkenyl” and “cycloalkenyl group” are used interchangeably and mean amonocyclic or polycyclic, unsaturated, non-aromatic ring comprisingcarbon and hydrogen atoms. Examples of cycloalkenyl groups include, butare not limited to, (C₃-C₁₀)cycloalkenyl groups, such as cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, andcyclooctenyl, and unsaturated cyclic and bicyclic terpenes. Acycloalkenyl group can be unsubstituted or substituted by one or twosuitable substituents. In one embodiment, the cycloalkenyl group is amonocyclic ring or bicyclic ring.

As used herein and unless otherwise indicated, the term“heterocycloalkenyl group” means a cycloalkenyl group as defined above,but with at least one heteroatom, for example, 1 to 3 heteroatomsselected from nitrogen, oxygen, and sulfur. A heterocycloalkenyl groupcan be unsubstituted or substituted with one or two suitablesubstituents. In one embodiment, the heterocycloalkenyl group is amonocyclic or bicyclic ring. In a more specific embodiment, theheterocycloalkenyl group is a monocyclic ring, wherein the ringcomprises from 3 to 8 carbon atoms and from 1 to 3 heteroatoms, referredto herein as “(C₃-C₈)heterocycloalkyl.”

As used herein and unless otherwise indicated, the terms “cyclicalkynyl” and “cycloalkynyl group” are used interchangeably and mean amonocyclic or polycyclic unsaturated ring comprising carbon and hydrogenatoms and having at least carbon-carbon triple bond. A cycloalkynylgroup can be unsubstituted or substituted by one or two suitablesubstituents. In one embodiment, the cycloalkynyl group is a monocyclicring or bicyclic ring.

As used herein and unless otherwise indicated, the term“heterocycloalkynyl group” means a cycloalkynyl group as defined above,but with at least one heteroatom, for example, 1 to 3 heteroatomsselected from nitrogen, oxygen, and sulfur. A heterocycloalkenyl groupcan be unsubstituted or substituted with one or two suitablesubstituents. In one embodiment, the heterocycloalkenyl group is amonocyclic or bicyclic ring. In a more specific embodiment, theheterocycloalkenyl group is a monocyclic ring, wherein the ringcomprises from 8 to 12 carbon atoms and from 1 to 3 heteroatoms,referred to herein as “(C₈-C₁₂)heterocycloalkynyl.”

As used herein and unless otherwise indicated, the term “heterocyclicradical” or “heterocyclic ring” means a heterocycloalkyl group or aheteroaryl group.

As used herein and unless otherwise indicated, the term “halogen” meansfluorine, chlorine, bromine, or iodine. Correspondingly, the meaning ofthe terms “halo” and “Hal” encompass fluoro, chloro, bromo, and iodo.

As used herein and unless otherwise indicated, the term “formulation”refers to a composition comprising a compound of the invention that isdescribed in a particular dosage form (e.g., tablet) or with aparticular dosage amount (e.g., 30 mg/kg).

As used herein and unless otherwise indicated, the term “hydrocarbylgroup” means a monovalent group selected from (C₁-C₈)alkyl,(C₂-C₈)alkenyl, and (C₂-C₈)alkynyl, optionally substituted with one ortwo suitable substituents. In one embodiment, the hydrocarbon chain of ahydrocarbyl group is from 1 to 6 carbon atoms in length, referred toherein as “(C₁-C₆)hydrocarbyl.”

When administered to a mammal (e.g., to an animal for veterinary use orto a human for clinical use), the compounds of the invention can beoptionally administered in isolated form. As used herein, “isolated”means that the compounds of the invention are at least partially removedfrom its natural or original environment, for example (a) a naturalsource, such as a plant or cell culture or (b) a synthetic organicchemical reaction mixture in which the compound is initially produced.For example, an isolated compound of the present invention as usedherein may still have residual components from the original chemicalreaction and not necessarily purified to any level. As used herein,“purified” means that when isolated, the isolate contains at least 80%,85%, 90%, 95%, 96%, 97%, 98% and 99% of the compound of the invention byweight.

The phrase “pharmaceutically acceptable salt(s),” as used hereinincludes but is not limited to salts of acidic or basic groups that maybe present in compounds used in the present compositions. Compoundsincluded in the present compositions that are basic in nature arecapable of forming a wide variety of salts with various inorganic andorganic acids. The acids that may be used to prepare pharmaceuticallyacceptable acid addition salts of such basic compounds are those thatform non-toxic acid addition salts, i.e., salts containingpharmacologically acceptable anions including, but not limited to,sulfuric, citric, maleic, acetic, oxalic, hydrochloride, hydrobromide,hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,isonicotinate, acetate, lactate, salicylate, citrate, acid citrate,tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds includedin the present compositions that include an amino moiety may formpharmaceutically acceptable salts with various amino acids, in additionto the acids mentioned above. Compounds included in the presentcompositions that are acidic in nature are capable of forming base saltswith various pharmacologically acceptable cations. Examples of suchsalts include alkali metal or alkaline earth metal salts and,particularly, calcium, magnesium, sodium lithium, zinc, potassium, andiron salts.

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable prodrug” means a derivative of a compoundthat can hydrolyze, oxidize, or otherwise react under biologicalconditions, in vitro or in vivo, to provide the compound. Examples ofprodrugs include, but are not limited to, compounds that comprisebiohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzableesters, biohydrolyzable carbamates, biohydrolyzable carbonates,biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Otherexamples of prodrugs include but are not limited to compounds thatcomprise oligonucleotides, peptides, lipids, aliphatic and aromaticgroups, or NO, NO₂, ONO, and ONO₂ moieties. Prodrugs can typically beprepared using well known methods, such as those described in Burger'sMedicinal Chemistry and Drug Discovery, pp. 172, 178, 949, 982 (ManfredE. Wolff ed., 5th ed. 1995), and Design of Prodrugs (H. Bundgaard ed.,Elselvier, New York 1985).

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide,”“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureide, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, lower acyloxyalkyl esters (such as acetoxylmethyl,acetoxyethyl, aminocarbonyloxy-methyl, pivaloyloxymethyl, andpivaloyloxyethyl esters), lactonyl esters (such as phthalidyl andthiophthalidyl esters), lower a lkoxyacyloxyalkyl esters (such asmethoxycarbonyloxy-methyl, ethoxycarbonyloxy-ethyl andisopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters,and acylamino alkyl esters (such as acetamidomethyl esters). Examples ofbiohydrolyzable amides include, but are not limited to, lower alkylamides, a amino acid amides, alkoxyacyl amides, andalkylaminoalkyl-carbonyl amides. Examples of biohydrolyzable carbamatesinclude, but are not limited to, lower alkylamines, substitutedethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic andheteroaromatic amines, and polyether amines.

As used herein and unless otherwise indicated, the phrase“therapeutically effective amount” of a composition of the invention ismeasured by the therapeutic effectiveness of a compound of theinvention, wherein at least one adverse effect of a disorder isameliorated or alleviated.

As set forth herein, the invention includes, but is not limited to,compounds, compositions and formulations for treating or preventingtreating or preventing a disease or disorder including, but not limitedto, conditions caused by uncontrolled cell growth, hyperproliferation ofcells, tumor growth, and cancers, for example, lung cancer, pancreaticcancer, leukemia, breast cancer, liver cancer, kidney cancer, ovariancancer, human glioblastoma and prostate cancer, which comprisesadministering to a mammal in need of such treatment or prevention atherapeutically or prophylactically effective amount of a compositioncomprising a compound of Formula I, II or III, or a pharmaceuticallyacceptable salt or prodrug thereof, and a pharmaceutically acceptablevehicle. In certain embodiments, a composition or formulation comprisinga compound of Formula I, II or III is useful in treating or preventingconditions caused by uncontrolled cell growth. In certain embodiments, acomposition or formulation comprising a compound of Formula I, II or IIIis useful in killing abnormal or cancerous cells while simultaneouslynot affecting healthy or normal cells. In certain embodiments, acomposition or formulation comprising a compound of Formula I, II or IIIact as cytotoxic agents. In certain embodiments, a composition orformulation comprising a compound of Formula I, II or III act asapoptotic agents. The compounds and compositions of Formulas I, II orIII can be used in ER-α positive cells and ER-α-negative cells. Inaddition, the compounds and compositions of Formulas I, II or III can beused in cells with a BRCA1 mutation and in cells without a BRCA1mutation.

In one embodiment, the present invention encompasses compounds andcompositions and formulations for treating abnormal cell growthcomprising contacting the cell or cells with at least one compound ofFormula I:

or pharmaceutically acceptable salts or prodrugs thereof, wherein

-   R₁, R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H; a halogen;    —OH; an amine; a carboxyl; substituted or unsubstituted: cyclic or    acyclic alkyl group, cyclic or acyclic alkenyl group, cyclic or    acyclic akynyl group, carboxyalkyl, cyclic or acyclic alkoxy, aryl    group, alkylaryl group, arylalkyl group, heteroaryl group,    heteroalkyl group; COR₁₀; CSR₁₀; SOR₁₀; SO₂R₁₀; CON(R₁₀)₂;    CSN(R₁₀)₂; wherein R₅ and R₆, or R₃ and R₄, or R₇ and R₈ can form a    3-10 membered substituted or unsubstituted ring with one or more of    C, S, O, or N,-   R₂ is CH₂, CHR₁, COR₁, CSR₁, SO, SO₂, SHOR₁, NH, NOR₁ or O,-   R₉ is CO, O, NH or CH₂, and-   R₁₀ is H; —OH; an amine; a carboxyl; substituted or unsubstituted:    cyclic or acyclic alkyl group, cyclic or acyclic alkenyl group,    cyclic or acyclic akynyl group, an aryl group, an alkylaryl group,    an arylalkyl group, heteroaryl group, heteroalkyl group; or a    halogen.

In one specific embodiment, the present invention encompasses compoundsand compositions and formulations for treating abnormal cell growthcomprising contacting the cell or cells with at least one compound ofFormula Ia below. Formula I herein includes but is not limited to allembodiments that are represented by Formula Ia below:

or pharmaceutically acceptable salts or prodrugs thereof, wherein

-   R₁, R₃, R₄, R₅ and R₆, are each independently H; a halogen; —OH; an    amine; a carboxyl; substituted or unsubstituted: cyclic or acyclic    alkyl group, cyclic or acyclic alkenyl group, cyclic or acyclic    akynyl group, carboxyalkyl, cyclic or acyclic alkoxy, aryl group,    alkylaryl group, arylalkyl group, heteroaryl group, heteroalkyl    group; COR₁₀; CSR₁₀; SOR₁₀; SO₂R₁₀; CON(R₁₀)₂; CSN(R₁₀)₂; wherein R₅    and R₆, or R₃ and R₄ can form a 3-10 membered substituted or    unsubstituted ring with one or more of C, S, O, or N,-   R₂ is CH₂, CHR₁, COR₁, CSR₁, SO, SO₂, SHOR₁, NH, NOR₁ or O,-   R₉ is CO, O, NH or CH₂, and-   R₁₀ is H; —OH; an amine; a carboxyl; substituted or unsubstituted:    cyclic or acyclic alkyl group, cyclic or acyclic alkenyl group,    cyclic or acyclic akynyl group, an aryl group, an alkylaryl group,    an arylalkyl group, heteroaryl group, heteroalkyl group; or a    halogen, and-   R₁₁ is CH₂, CHR₁ or NR₁.

Illustrative specific embodiments of compounds of the present inventionuseful in the methods for treating abnormal cell growth include but arenot limited to compounds 101-114 disclosed below.

With respect to the captioned structures above, the present inventionprovides specific embodiments wherein R₁ is F, Cl, Br, I, H, CH₃ orBenzyl and R₂ is CH₂, NH or O, as listed below. For example, forcompounds 101-112 disclosed above, specific embodiments are as follows:

a, R₁ is F and R₂ is CH₂; b, R₁ is F and R₂ is NH; c, R₁ is F and R₂ isO; d, R₁ is Cl and R₂ is CH₂; e, R₁ is Cl and R₂ is NH; f, R₁ is Cl andR₂ is O; g, R₁ is Br and R₂ is CH₂; h, R₁ is Br and R₂ is NH; i, R₁ isBr and R₂ is O; j, R₁ is I and R₂ is CH₂; k, R₁ is I and R₂ is NH; m, R₁is I and R₂ is O; n, R₁ is H and R₂ is CH₂; o, R₁ is H and R₂ is NH; p,R₁ is H and R₂ is O; q, R₁ is CH₃ and R₂ is CH₂; r, R₁ is CH₃ and R₂ isNH; s, R₁ is CH₃ and R₂ is O; t, R₁ is Benzyl and R₂ is CH₂; u, R₁ isBenzyl and R₂ is NH; v, R₁ is Benzyl and R₂ is O;

From the disclosed generic structures and the chart above, examples ofspecific embodiments of the present invention such as 101a and 103mwould be represented by the chemical formulas

Likewise, compounds 109c and 109h would be represented as follows:

In another embodiment, the present invention encompasses compounds andcompositions and formulations for treating abnormal cell growthcomprising contacting the cell or cells with at least one compound ofFormula II:

or pharmaceutically acceptable salts or prodrugs thereof, wherein

-   R₁ is H; a halogen; —OH; an amine; a carboxyl; substituted or    unsubstituted: cyclic or acyclic alkyl group, cyclic or acyclic    alkenyl group, cyclic or acyclic akynyl group, carboxyalkyl, cyclic    or acyclic alkoxy, aryl group, alkylaryl group, arylalkyl group,    heteroaryl group, heteroalkyl group; COR₈; CSR₈; SOR₈; SO₂R₈;    CON(R₈)₂; CSN(C₈)₂; wherein R₁ and R₄ can form a 3-10 membered ring    with one or more of C, S, O or N;-   R₂ and R₅ are each independently H; a halogen; —OH; an amine; a    carboxyl; substituted or unsubstituted: cyclic or acyclic alkyl    group, cyclic or acyclic alkenyl group, cyclic or acyclic akynyl    group, carboxyalkyl, cyclic or acyclic alkoxy, aryl group, alkylaryl    group, arylalkyl group, heteroaryl group, heteroalkyl group; COR₈;    CSR₈; SOR₈; SO₂R₈; CON(R₈)₂; CSN(R₈)₂; or a monocyclic or polycyclic    substituted or unsubstituted fused ring structure that is fused with    the ring atoms to which either R₂ or R₅ are attached; wherein R₂ and    R₅ can form a 3-10 membered ring with one or more of C, S, O or N;-   R₃ and R₄ are each independently CH; C(R₁)₂; CO; COR₁; CON(R₁)₂; CS;    CSR₁; CS(R₁)₂; C(SR₁)R₁; CSNR₁; SH; SO; SO₂; S(O)R₁; S(O)(R₁)₂; N;    NH; NR₁; NOR₁; or O; wherein R₃ and R₄, together with the ring atoms    to which R₄ is attached, can form an additional fused 5- or    6-membered ring structure, with one or more of C, S, O or N; wherein    R₂ and R₃ together with the ring atoms to which they are attached,    can form an additional fused 5- or 6-membered ring structure, with    one or more of C, S, O or N; wherein R₃ together with the ring atoms    to which it is attached, can form an additional fused 5- or    6-membered ring structure, with one or more of C, S, O or N;-   R₈ is H; —OH; an amine; a carboxyl; substituted or unsubstituted:    cyclic or acyclic alkyl group, cyclic or acyclic alkenyl group,    cyclic or acyclic akynyl group, an aryl group, an alkylaryl group,    an arylalkyl group, heteroaryl group, heteroalkyl group; or a    halogen; and-   is a single bond or a double bond, wherein if    is a double bond, R₃ and R₄ are independently CH, N or SH.

Illustrative embodiments of compounds of the present invention useful inmethods for treating abnormal cell growth include but are not limited tocompounds 201-216 as disclosed below.

In another embodiment, the present invention encompasses compounds andcompositions and formulations for treating abnormal cell growthcomprising contacting the cell or cells with at least one compound ofFormula III:

or pharmaceutically acceptable salts or prodrugs thereof, wherein

-   R₁ and R₃ are each independently H; a halogen; —OH; an amine; a    carboxyl; substituted or unsubstituted: cyclic or acyclic alkyl    group, cyclic or acyclic alkenyl group, cyclic or acyclic alkynyl    group, carboxyalkyl, cyclic or acyclic alkoxy, aryl group, alkylaryl    group, arylalkyl group, heteroaryl group, heteroalkyl group; COR₈;    CSR₈; SOR₈; SO₂R₈; CON(R₈)₂; CSN(R₈)₂; wherein R₁ and R₃ can form a    6-15 membered ring with one or more of C, S, O, or N,-   R₂ and R₄ are each independently H; a halogen; —OH; an amine; a    carboxyl; substituted or unsubstituted: cyclic or acyclic alkyl    group, cyclic or acyclic alkenyl group, cyclic or acyclic akynyl    group, carboxyalkyl, cyclic or acyclic alkoxy, aryl group, alkylaryl    group, arylalkyl group, heteroaryl group, heteroalkyl group; COR₈;    CSR₈; SOR₈; SO₂R₈; CON(R₈)₂; CSN(R₈)₂; wherein R₂ and R₄ can form a    monocyclic or polycyclic 3-14 membered fused ring structure,    together with the atoms to which they are attached, with one or more    of C, S, O, or N, and-   R₈ is —H; —OH; an amine; a carboxyl; substituted or unsubstituted:    cyclic or acyclic alkyl group, cyclic or acyclic alkenyl group,    cyclic or acyclic akynyl group, an aryl group, an alkylaryl group,    an arylalkyl group, heteroaryl group, heteroalkyl group; or a    halogen.

Illustrative embodiments of compounds of the present invention useful inmethods for treating abnormal cell growth include but are not limited tocompounds 301-307 and 308-314 as disclosed below.

With respect to the compounds of 308, the present invention providesspecific embodiments wherein R₁, R₂ and R₃, are each independently NH₂,OH, OCH₃, Cl, Br, CH₃, CH(CH₃)₂, SO₂NH₂, COOH, CH₂CH₃ or CF₃, as listedbelow.

With respect to the compounds of 308, the present invention providesspecific embodiments wherein R₁, R₂ and R₃, are each independently NH₂,OH, OCH₃, Cl, Br, CH₃ or CF₃. Additional embodiments of the presentinvention include compounds 309 and 310 below.

In select embodiments, the novel compounds of Formulas I, II and III donot encompass compounds 103f, 201, 301, 305 and 901-922 as thesecompounds that are explicitly excluded from the scope of novel compoundsunder Formulas I, II and III. All compounds of the present inventionincluding compounds 103f, 201, 301, 305 and 901-922, however, can beused in the novel and inventive methods of treating abnormal cell growthas disclosed in the present invention.

The compounds of the present invention can be synthesized by organicchemistry techniques known to those skilled in the art, as generallydescribed by, but not limited to, the Schemes below.

Scheme 1—Rapid access to compounds of Formula I with two of the fourpossible stereochemical arrangements of the cyclopropane ring can beobtained by dicyclocarbodiimide-mediated coupling of 6-chloropiperonylicacid (3) with trans-chrysanthemyl alcohol (2a) or chrysanthemyl alcohol(2a) (below). To obtain other stereochemical arrangements of thecyclopropane ring system, however, Charette's catalyst (10) can beutilized for asymmetric cyclopropanation of mono-TBS(t-butyldimethylsilyl) protected 2-butenediol (4). Oxidation of thealcohol with TPAP (tetra-n-propylammonium perruthenate) followed byisopropylidene Wittig reagent and deprotection of the TBS group withTBAF (tetrabutylammonium fluoride) will yield the requisite alcohol (6)for coupling with 6-chloropiperonylic acid (3). The cis-cyclopropanesystem can be synthesized by an analogous route from mono-TBS protectedZ-2-butenediol (7). Asymmetric cyclopropanation with Charette's catalyst(10) will give compound (8). TPAP oxidation, Wittig homologation, andTBAF-deprotection of the TBS group will give alcohol (9), which can alsobe conjugated with 6-chloro or 6-methyl piperonylic acid.

Finally, to incorporate the 1,1-dimethyl cyclopropane moiety with fullstereocontrol, Evans' isopropyl chiral oxazolidinone auxiliary will beconjugated to senecioic acid to give compound (12). Rhodiummediatedcyclopropanation with methyl diazoacetate should give a mixture ofdiastereomers (13), where the chiral auxiliary controls the facialselectivity of cyclopropanation, but the carbomethoxy group can be cisor trans to the carbonyl of the auxiliary, which should be a mixtureseparable by chromatography. Selective saponification of the methylester can be followed by TPAP oxidation to the aldehyde, Wittighomologation, and DIBAL-H (diisobutylaluminum hydride) reduction of thechiral auxiliary to the alcohol, to give compound (14) or (15).

Scheme 2—Compound (17) can be synthesized via a malonic ester synthesisfrom the available 2-bromomethyl-naphthalene (16) below. The2-(2-iodo-ethyl)-naphthalene (18) is added to the dihydro-furan-2-one(19) to give compound (20). Using “click chemistry” the triazole (22) issynthesized from the azide (21). The acid (24) can be made from the2-bromomethyl-biphenyl 23 via a malonic ester synthesis. Horner-Emmonschemistry is used to convert the ketone (25) into the(3,4-dihydro-1H-anthracen-2-ylidene)-acetic acid (26). Hydrogenation of(26) can provide one compound of the present invention (compound 204).

Scheme 3—The synthesis of symmetric analogs of 4-aminopyrimidines viatrimerization of nitriles using microwave conditions is employed asdescribed before (Baxendale, I.R., et al., J. Combin. Chem., 7:483-489(2005)) and is incorporated by reference. To synthesize the proposedasymmetric aminopyrimidines, substituted benzamidines (30) are reactedwith substituted 3-oxo-butyric acid methyl esters to give2-phenyl-pyrimidin-4-ols (32). Analogs like (32) can be converted intothe final substituted 4-aminopyrimidine (34). Alternatively, the4-amino-2,6-dichloro-pyrimidine-5-carbonitrile (36) can be synthesizedfrom 6-amino-2,4-dioxo-1,2,3,4-tetrahydro-pyrimidine-5-carbonitrile(35). Finally, the restricted analog (41) is made via a reaction of theazido-benzamidine (37) and the ester (38). Click chemistry affords thetriazole (40) and is followed by conversion of (40) to theaminopyrimidine (41).

The compounds and compositions according to Formulas I, II or III of thepresent invention are useful in treating abnormal cell growth. In oneembodiment, the abnormal cell growth is cancer or cancer cell growth orproliferation. As used herein and unless otherwise indicated, the terms“cancer” or “cancer cell” refer to abnormal cell growth or proliferationthat may or may not include spontaneous or induced phenotypic changes.As used herein, “cancer” includes but is not limited to such abnormalconditions as hypertrophy, neoplasia, hyperplasia, benign and malignantcancer. As used herein, the term “tumor” is a general term that includeshypertrophies, neoplasias, hyperplasias, benign cancers and malignantcancers. Accordingly, certain embodiments of the present inventioninclude but are not limited to treating a hypertrophy, a neoplasia, ahyperplasia, a benign or a malignant cancer in a subject. In additionalembodiments, the present invention is directed to preventing or reducingthe likelihood of metastasis and/or recurrence of a hypertrophy, aneoplasia, a hyperplasia, a benign or a malignant cancer within asubject comprising administering at least one compound of the presentinvention to the subject. For example, at least one compound of thepresent invention may be administered after tumorresection/removal/ablation, etc. to reduce the likelihood of recurrenceof the tumor in the subject. In another example, at least one compoundof the present invention may be administered to reduce the likelihood ofmetastasis of the tumor in the subject.

These methods include but are not limited to inhibiting or reducing thegrowth of a cancer cell or cells, such as lung cancer cell(s), breastcancer cell(s), colon cancer cell(s), malignant melanoma cell(s),ovarian carcinoma cell(s), brain tumor cell(s), soft tissue sarcomacell(s), rhabdomyosarcoma cell(s), pancreatic cancer cell(s), prostatecancer cell(s) and osteosarcoma cell(s), which comprises administeringto the cell a pharmaceutically effective amount of a compositioncomprising a compound of Formula I, II or III or a pharmaceuticallyacceptable salt or prodrug thereof.

The invention also encompasses a method of reducing the likelihood ofcancer metastasis, such as lung cancer metastasis, breast cancermetastasis, colon cancer metastasis, malignant melanoma metastasis,ovarian carcinoma metastasis, brain tumor metastasis, soft tissuesarcoma metastasis, rhabdomyosarcoma metastasis, pancreatic cancermetastasis, prostate cancer metastasis and osteosarcoma metastasis,which comprises administering to a subject in need of such treatment atherapeutically effective amount of a composition comprising a compoundof Formula I, II or III or a pharamaceutically acceptable salt orprodrug thereof.

In one embodiment, “treatment” or “treating” refers to an ameliorationof a disease or disorder, or at least one detectable symptom thereof. Inanother embodiment, “treatment” or “treating” refers to an ameliorationof at least one measurable physical parameter, not necessarilydiscernible by the patient. In yet another embodiment, “treatment” or“treating” refers to inhibiting the progression of a disease ordisorder, either physically, e.g., stabilization of a discerniblesymptom, physiologically, e.g., stabilization of a physical parameter,or both. In yet another embodiment, “treatment” or “treating” refers todelaying the onset of a disease or disorder.

In certain embodiments, the compositions of the invention areadministered to a patient, for example a human, as a preventativemeasure against diseases, including preventing the occurrence orreoccurrence of a tumor or preventing or slowing the progression of atumor.

As used herein, the term “prevent,” as it relates to tumors and/orabnormal cell growth, indicates that a compound of the present inventionis administered to a subject to at least partially inhibit the or reducethe likelihood of growth, division, spread, or proliferation of tumorcells. Thus a subject may be “pretreated,” by administering the one ormore compounds of the present invention to prevent tumors from arising.The phrase “preventing the progression,” as it relates to tumors, isused to mean a procedure designed to at least partially inhibit thedetectable appearance of one or more additional tumors or aberrant cellgrowth in a patient already exhibiting one or more symptoms of thepresence of a tumor or aberrant cell growth, and is also used to mean atleast partially prohibiting the already-present symptoms of cancer fromworsening in the subject.

The compounds or compositions of the present invention can be used totreat abnormal cell growth by contacting the cell or cells with at leastone compound or composition of the present invention. As used herein,the term “contact” when used with respect to when the compounds orcompositions of the present invention are being used for the methodsdisclosed herein refers to the act of bringing the compounds orcompositions in proximity with one another such that the compounds orcompositions can exert their biological effects on the cell or cells.Bringing the compounds or compositions into contact with a cell or cellscan mean applying the compounds or compositions directly onto the cellssuch as in an in vitro setting. Alternatively, bringing the compounds orcompositions into contact with a cell or cells can mean administeringthe compounds or compositions to a subject or patient.

As used herein, the term “administer” and “administering” are used tomean introducing at least one compound or composition into a subject.When administration is for the purpose of treatment, the substance isprovided at, or after the diagnosis of an abnormal cell growth, such asa tumor. The therapeutic administration of this substance serves toinhibit cell growth of the tumor or abnormal cell growth.

As used herein, the term “coadminister” is used to mean that each of atleast two compounds are administered during a time frame wherein therespective periods of biological activity overlap. Thus the termincludes sequential as well as coextensive administration of thecompositions of the present invention. If more than one substance iscoadministered, the routes of administration of the two or moresubstances need not be the same. The scope of the invention is notlimited by the identity of the substance which may be coadministeredwith the compositions of the present invention. For example, one of thecompounds of the present invention may be co-administered with anothercompound of the present invention or another other pharmaceuticallyactive substances, such as vinca alkaloids, nucleic acid inhibitors,platinum agents, interleukin-2, interferons, alkylating agents,antimetabolites, corticosteroids, DNA intercalating agents,anthracyclines, and ureas. Examples of specific agents in addition tothose exemplified herein, include hydroxyurea, 5-fluorouracil,anthramycin, asparaginase, bleomycin, dactinomycin, dacabazine,cytarabine, busulfan, thiotepa, lomustine, mechlorehamine,cyclophosphamide, melphalan, mechlorethamine, chlorambucil, carmustine,6-thioguanine, methotrexate, etc.

Due to the activity of the compounds of the invention, the compounds areadvantageously useful in veterinary and human medicine. As describedabove, the compounds of the invention are useful for the treatment orprevention of conditions caused by uncontrolled cell growth,hyperproliferation of cells, tumor growth, and cancers, for example,lung cancer, pancreatic cancer, leukemia, breast cancer, liver cancer,kidney cancer, human glioblastoma and prostate cancer.

The invention provides methods of treatment and prophylaxis byadministration to a subject of a therapeutically effective amount of acomposition comprising a compound of the invention. The subject can be amammal, including, but is not limited to, an animal such a cow, horse,sheep, pig, chicken, cat, dog, mouse, rat, rabbit, guinea pig, non-humanprimate or human.

The present compositions, which comprise one or more compounds of theinvention can be administered intravenously, intravenouslyintramuscularly intraperitonealy and orally.

Suitable dosage ranges of the compounds of the invention, regardless ofthe route of administration, are generally about 0.0001 milligram to2000 milligrams of a compound of the invention per kilogram body weight.In one specific embodiment, the dose is about 0.001 milligram to about1500 milligrams per kilogram body weight, more specifically about 0.01milligram to about 1000 milligrams per kilogram body weight, morespecifically about 0.1 milligram to about 500 milligrams per kilogrambody weight, and yet more specifically about 1 milligram to about 100milligrams per kilogram body weight.

The compounds and the compositions of the invention may also beadministered by any other route, for example, by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.), and they may beadministered together with another biologically active agent.Administration can be systemic or local. Various delivery systems areknown, e.g., encapsulation in liposomes, microparticles, microcapsules,capsules, etc., and can be used to administer a compound or compositionof the invention. In certain embodiments, more than one compound of theinvention is administered to a patient. Methods of administrationinclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral,sublingual, intranasal, intracerebral, intravaginal, transdermal,rectally, by inhalation, or topically. The preferred mode ofadministration is left to the discretion of the practitioner, and willdepend in-part upon the site of the medical condition.

In specific embodiments, it may be desirable to administer one or morecompounds or compositions of the invention locally to the area in needof treatment. This may be achieved, for example, and not by way oflimitation, by local infusion during surgery, topical application, e.g.,in conjunction with a wound dressing after surgery, by injection, bymeans of a catheter, by means of a suppository, or by means of animplant, said implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as but not limited to silasticmembranes, or fibers. In one embodiment, administration can be by directinjection at the site (or former site) of an atherosclerotic plaquetissue.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Incertain embodiments, the compounds of the invention can be formulated asa suppository, with traditional binders and vehicles such astriglycerides.

The present compositions will contain a therapeutically effective amountof a compound of the invention, optionally more than one compound of theinvention, preferably in purified form, together with a suitable amountof a pharmaceutically acceptable vehicle so as to provide the form forproper administration to the patient.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “vehicle” refers to a diluent, adjuvant, excipient, or carrier withwhich a compound of the invention is administered. Such pharmaceuticalvehicles can be liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. The pharmaceuticalvehicles can be saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea, and the like. In addition, auxiliary,stabilizing, thickening, lubricating and coloring agents may be used.When administered to a patient, the compounds of the invention andpharmaceutically acceptable vehicles are preferably sterile. Water is apreferred vehicle when the compound of the invention is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid vehicles, particularly forinjectable solutions. Suitable pharmaceutical vehicles also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The present compositions, if desired, canalso contain minor amounts of wetting or emulsifying agents, or pHbuffering agents.

The present compositions can take the form of solutions, suspensions,emulsion, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,emulsions, aerosols, sprays, suspensions, or any other form suitable foruse.

In another embodiment, the compounds and/or compositions of theinvention are formulated in accordance with routine procedures as apharmaceutical composition adapted for intravenous administration tohuman beings. Typically, compounds and/or compositions of the inventionfor intravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the compositions may also include asolubilizing agent. Compositions for intravenous administration mayoptionally include a local anesthetic such as lignocaine to ease pain atthe site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the compound of the invention is to beadministered by infusion, it can be dispensed, for example, with aninfusion bottle containing sterile pharmaceutical grade water or saline.Where the compound of the invention is administered by injection, anampoule of sterile water for injection or saline can be provided so thatthe ingredients may be mixed prior to administration.

In one specific embodiment, the compositions of the invention can beadministered orally. Formulations for oral delivery may be in the formof tablets, lozenges, aqueous or oily suspensions, granules, powders,emulsions, capsules, syrups, or elixirs, for example. Orallyadministered compositions may contain one or more optionally agents, forexample, sweetening agents such as fructose, aspartame or saccharin;flavoring agents such as peppermint, oil of wintergreen, or cherry;coloring agents; and preserving agents, to provide a pharmaceuticallypalatable preparation. Moreover, where in tablet or pill form, thecompositions may be coated to delay disintegration and absorption in thegastrointestinal tract thereby providing a sustained action over anextended period of time. Selectively permeable membranes surrounding anosmotically active driving compound are also suitable for orallyadministered compounds of the invention. In one particular platform,fluid from the environment surrounding the capsule is imbibed by thedriving compound, which swells to displace the agent or agentcomposition through an aperture. These delivery platforms can provide anessentially zero order delivery profile as opposed to the spikedprofiles of immediate release formulations. A time delay material suchas glycerol monostearate or glycerol stearate may also be used. Oralcompositions can include standard vehicles such as mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, etc. Such vehicles are preferably of pharmaceutical grade.

The amount of a compound of the invention that will be effective in thetreatment of a particular disorder or condition disclosed herein willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro or in vivo assaysmay optionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the compositions will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. In specific embodiments of the invention,the oral dose of at least one compound of the present invention is about0.01 milligram to about 100 milligrams per kilogram body weight, or fromabout 0.1 milligram to about 50 milligrams per kilogram body weight, orfrom about 0.5 milligram to about 20 milligrams per kilogram bodyweight, or from about 1 milligram to about 10 milligrams per kilogrambody weight.

Suitable dosage ranges for parenteral, for example, intravenous (i.v.)administration are 0.01 milligram to 100 milligrams per kilogram bodyweight, 0.1 milligram to 35 milligrams per kilogram body weight, and 1milligram to 10 milligrams per kilogram body weight. Suitable dosageranges for intranasal administration are generally about 0.01 pg/kg bodyweight to 1 mg/kg body weight. Suppositories generally contain 0.01milligram to 50 milligrams of a compound of the invention per kilogrambody weight and comprise active ingredient in the range of 0.5% to 10%by weight. Recommended dosages for intradermal, intramuscular,intraperitoneal, subcutaneous, epidural, sublingual, intracerebral,intravaginal, transdermal administration or administration by inhalationare in the range of 0.001 milligram to 200 milligrams per kilogram ofbody weight. Suitable doses of the compounds of the invention fortopical administration are in the range of 0.001 milligram to 1milligram, depending on the area to which the compound is administered.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems. Such animal models andsystems are well known in the art.

In other embodiments, a composition of the invention for parenteral, forexample, intravenous administration includes about 0.001 milligram toabout 2000 milligrams of a compound of the invention, more preferablyabout 0.01 milligram to about 1000 milligrams of a compound of theinvention, more preferably about 0.1 milligram to about 500 milligramsof a compound of the invention, and yet more preferably about 1milligram to about 200 milligrams of a compound of the invention.

The invention also provides pharmaceutical packs or kits comprising oneor more containers filled with one or more compounds of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In a certain embodiment, the kit contains more than onecompound of the invention.

The compounds of the invention can be assayed in vitro and in vivo, forthe desired therapeutic or prophylactic activity, prior to use inhumans. For example, in vitro assays can be used to determine whetheradministration of a specific compound of the invention or a combinationof compounds of the invention can be used for treating a particulardisorder or condition disclosed herein. The compounds of the inventionmay also be demonstrated to be effective and safe using animal modelsystems.

Other methods will be known to the skilled artisan and are within thescope of the invention.

EXAMPLES Example 1

Purified recombinant proteins BRCA1 (1-100), ER-α (337-379), ER-α(282-420), and ER-α (421-595) using various tags (GST, His, andThioredoxin) have been successfully produced. BRCA1 (101-200), BRCA1(1-200), and ER-α (282-595) can also be produced.

Surface Plasmon Resonance (SPR)-based biophysical assays (Biacore) ofthe compounds of the present invention can be used to assess real-timekinetics of molecular interaction. Drug screening can be performeddirectly from 96-well plates seeded with any of the compounds of thepresent invention. First, an SPR study will be used to assess theinteraction of BRCA1 and ER-α and determine which fragments of eachprotein are best suited for the Biacore screening study. The bindingsites of BRCA1 have been mapped to the N-terminus of BRCA1 (within aa1-100 and secondarily aa 101-200) and the AF-2/LBD region of ER-α (aa282-420 with some contributions from aa 421-595). BRCA1 (1-100, 101-200,and 1-200), and ER-α (282-420 and 282-595) are used to confirm thephysical interaction of the fragments of the two proteins and determinethe kinetics using Biacore. A combination of two fragments that bestmaintain the high affinity BRCA1: ER-α interaction is selected forscreening.

Example 2

To determine the effects of test compounds on cathepsin D and pS2expression, MCF-7 or T47D cells are cultured in DMEM containing 5%charcoal- stripped serum (CSS) and are treated with and withoutestradiol (E2) (10 nM) in the presence of either vehicle (DMSO) only,test compounds or known inactive compounds for about 24 hours andharvested for RNA and protein assays. Cathepsin D and pS2 mRNA levelsare determined by quantitative qRT-PCR; and the protein levels will bedetermined by Western blotting. As a positive control for inhibition ofcathepsin D and pS2, cells are transfected with wtBRCA1 or empty pcDNA3vector and then tested for E2-stimulated cathepsin D and pS2 expression.The BRCA1 and ER-α protein levels are monitored by Western blotting.

MCF-7 cells are first incubated in DMEM containing 5% CSS for 72 hours,with daily washing with fresh medium to remove E2 that may be stuck tocells. Cells are then treated with or without E2 at several differenttime points, e.g., 15, 30, 60, and 120 min, in the presence of a testcompound, a known inactive compound or vehicle (DMSO) and subsequentlyharvested for chromatin immunoprecipitation (ChIP) assays to assess thecontent of ER-α and BRCA1 at the estrogen response element (ERE) of thecathepsin D promoter. As a negative control, assays are carried outusing PCR primers corresponding to a region of the cathepsin D promoterdistant from the ERE. For comparison, similar experiments are performedon cells transfected with wtBRCA1 or pcDNA3 vector. The total cellularlevels of ER-α and BRCA1 are monitored by Western blotting.

Example 3

Another possible application of the compounds of the present inventionis in combination with other anti-estrogens (or other agents such aschemotherapy drugs or signal transduction inhibitors), particularly ifthe combinations of agents act synergistically but have differenttoxicities. An example might be a combination of BRCA1-mimetic plusTamoxifen to overcome the pro-estrogenic activity of Tamoxifen in theendometrium.

Tamoxifen alone stimulates ERE- TK-Luc reporter activity in MCF-7 cells,while it inhibits estradiol (E2)-stimulated reporter activity. Knockdownof BRCA1 shifts the balance of Tamoxifen activity from that of an ER-αantagonist towards that of an agonist, suggesting that BRCA1 regulatesthe antagonist vs. agonist properties of Tamoxifen.

Briefly, MCF7 or T47D cells are transfected overnight with an ERE-TK-Lucreporter construct. An ERE-TK-Luc construct contains an estrogenresponse element (ERE) upstream of a minimal thymidine kinase (TK)promoter that drives the expression of the luciferase (Luc) reportergene. After transfection, the cells are subsequently washed and allowedto recover for several hours. The cells are then incubated with andwithout E2 (at about 10 nM) along with the agent(s) to be tested for 24hours. The agents and combinations to be tested are: group 1) vehicle;group 2) test compound; group 3) Tamoxifen; group 4) Fulvestrant; group5) test compound+Tamoxifen; and group 6) test compound+Fulvestrant. Thecells are subsequently harvested for luciferase reporter assays. Theend-points are the ability of the combination (groups 5 and 6) toinhibit E2-stimulated reporter activity compared to each agent alone.Different concentrations of each agent are tested, with the goal ofdocumenting synergy for potentiation or inhibition of E2-stimulatedreporter activity (if it occurs) by showing that the test compound at aconcentration that is ineffective by itself can potentiate the ER-αinhibitory activity of Tamoxifen or Fulvestrant (or vice versa), and/orthat combinations of the test compound and Tamoxifen or Fulvestrant atconcentrations for which each agent is ineffective by itself cansignificantly inhibit E2-stimulated ER-α activity.

If a combination of a test compound plus Tamoxifen or Fulvestrant cansynergistically inhibit E2-stimulated ER-α activity, this combinationcan also be tested to inhibit E2-stimulated MCF-7 cell proliferation.

Example 4

Three compounds were tested for their ability to inhibit ER-α activity.Briefly, compounds 103(f), 201 and 301 were tested for their ability toinhibit E2-stimulated ER-α transcriptional activity in MCF-7 cellsutilizing the ERE-TK-Luc reporter to obtain a readout of ER-α activity.This has proved to be a reliable and reproducible assay of ER-α activityin many prior studies. A dose of 10 nM of E2 (17β-estradiol) wasutilized to stimulate ER-α activity. This dose yielded about a20-150-fold increase in ERE-TK-Luc activity, and a wtBRCA1 expressionvector reduced the E2-stimulated ER-α activity by ≥99%, as compared withempty pcDNA3 vector or a sham transfection control. Initially, eachcompound was tested at doses of 1 μM and 50 μM.

The values of IC₅₀ and IC₈₀ (concentrations required for inhibition ofER-α activity by 50% and 80%, respectively) are summarized in the Tablebelow.

Compound IC₅₀ (μM) IC₈₀ (μM) 103(f) 3 5 301 12 40 201 4 50

Like breast cancer, endometrial cancer development is E2-dependent, andwomen that carry BRCA1 mutations exhibit a significantly increased riskfor endometrial cancer. For reasons that are not well understood,Tamoxifen acts as an ER-α agonist in the uterus, where it stimulates thedevelopment of endometrial hyperplasia and carcinoma. Compounds 103(f)and 301 were tested to determine if they antagonize E2-stimulated ER-αactivity in endometrial cancer cells as they do in breast cancer cells.Three endometrial carcinoma cell lines (CRL2923, CRL1671, and CRL1622),all of which were obtained from the American Type Culture Collection,were tested. One of these cell lines is ER-positive (CRL2923), while theother two are ER-negative. For the two ER-negative cell lines, awild-type ER-α expression vector was co-transfected along with theERE-TK-Luc reporter to allow measurement of E2-stimulated ER-αtranscriptional activity. In all three cell lines tested, compounds103(f) and 301 each effectively inhibited E2-stimulated ER-α activity,with residual activity levels ranging from 11-25% at a concentration of50 μM (P<0.001). In these assays, E2-stimulated ER-α activity in theabsence of drug ranged from about 30-fold to about 80-fold, indicating arobust stimulation of ER-α activity similar to that obtained in breastcarcinoma cells at an E2 concentration of 10 nM.

Compound 103(f) was tested for its effect on E2 stimulated proliferationof MCF-7 cells. Briefly, cells were inoculated into 12-well dishes at1×10⁴ cells per well on day 0 in 5% charcoal-stripped serum (CSS). Onday 1, after allowing for cell attachment, the cells were refed freshmedium containing 2% CSS with or without estradiol (E2) (10 nM) andeither with vehicle (DMSO), 103(f) (5 μM or 20 μM). Triplicate wellswere counted on days 1-4 to determine the effect of 103(f) on cellgrowth. Cells treated with DMSO only continued to grow slowly andreached a density of about 4×10⁴ cells per well by day 4, while cellstreated with (DMSO+E2) grew much more rapidly and reached 9×10⁴ cellsper well by day 4 (P<0.01, two-tailed t-test). Compound 103(f) alone (5μM or 20 μM) had little or no effect on cell growth, as compared withDMSO, but in the presence of E2, compound 103(f) at either dose blockedmost of the E2-stimulated cell growth (P<0.01).

Example 5

A number of cell lines have been developed to investigate the mechanismsof anti-estrogen resistance. MCF-7/5-23, GI-101A, ZR-75-9a1, LY2, LCC2,and LCC9 are well-known examples of ER-α+ breast cancer cell lines thatare resistant to Tamoxifen and in some cases, other anti-estrogens,e.g., Fulvestrant. In some cell lines, e.g., LCC9, knockdown of ER-αusing siRNA inhibits cell proliferation, suggesting that ER-αcontributes to cell proliferation even though the cells areTamoxifen-resistant. To determine if the compounds of the presentinvention are active in cell lines that are resistant to Tamoxifen orFulvestrant, a set of MCF-7 derived cell lines with different E2 andanti-estrogen sensitivities can be used. For example, some cell linesare listed below:

Cell ER-α Response to Response to Line Status Tamoxifen FulvestrantResponse to Estradiol (E2) MCF-7 + Sensitive Sensitive E2-responsive;E2-dependent growth LCC1 + Sensitive Sensitive Partially E2-depenedentgrowth LCC2 + Resistant Sensitive E2-dependent growth LCC9 + ResistantResistant E2-insensitive growth; some E2-responsive components

LCC1, LCC2, and LCC9 are routinely maintained in phenol red-free DMEMcontaining 5% CSS (available from the Tissue Culture Core, LombardiCancer Center, Georgetown University Medical Center). MCF-7 (original)cells are cultured under normal conditions in DMEM containing 5% fetalcalf serum. To test the effects of various compounds of the presentinvention on the growth of LCC1/2/9, cells are seeded into 6-well dishesin phenol red-free DMEM with 5% CSS, allowed to attach for 24 hours andincubated with the test compound(s) for up to 6 days. MCF-7 cells arepre-incubated for at least 72 hours in phenol red-free DMEM with 5% CSSwith daily washing prior to addition of compounds. Triplicate wells arecounted on days 0, 2, 4, and 6, and each experiment is performed atleast three times for reproducibility. The agents to be tested include:(1) vehicle only (DMSO), (2) test compound (5 and 20 μM), (3) knowninactive compound, (4) test compound in (2) and E2 (10 nM), and (5)known inactive compound in (3) and E2. Other known anti-estrogens, e.g.,Tamoxifen (1 μM) or Fulvestrant (100 nM), can be used as control on theeffects of the test compounds on cell proliferation.

Example 6

A major function of BRCA1 is to promote error-free DNA double-strandbreak (DSB) repair by homologous recombination. The effect of testcompounds of the present invention on DSB repair can be tested bytransfection of a plasmid [HR-EGFP/3′- EGFP] that results in EGFPproduction when activated by DSB repair via homologous recombination.MCF-7 cells are co-transfected with wtBRCA1 or pcDNA3 vector todetermine the effect of the compounds on basal and BRCA1-stimulated DSBrepair. The cells are then incubated with test compounds at variousconcentrations or DMSO for about 3 or 4 days to determine the effect ofthe compound on the recombination frequency.

BRCA1 over-expression protects cells against, and its knockdownsensitizes cells to, oxidative stress in part by stimulating theactivity of the antioxidant transcription factor NFE2L2. Test compoundsare tested for their capacity to interfere with the ability of wtBRCA1to protect MCF-7 or T47D cells against oxidants (H₂O₂ and paraquat),using MTT assays to quantify cytotoxicity. The compounds can also betested for their ability to inhibit wtBRCA1 stimulation of NFE2L2activity using the NQ01-ARE-Luc reporter to measure NFE2L2 activity viathe antioxidant response element (ARE).

Example 7

Dose range of the test compounds can be based on solubility, which canaffect the choice of vehicle, route, volume, and maximum achievable dosefor each compound. Generally, intraperitoneal (ip) is a first choice asa route of administration, as the total volume (and hence dose)deliverable is greatest. A subcutaneous (sc) depot or intravenous (iv)injection are alternatives. All drug preparations are sterilized byfiltration prior to delivery. For aqueous soluble drugs,phosphate-buffered physiological saline at pH 7.4 is a standard vehicle.Ethanol and DMSO are used for less water-soluble agents, and cremaphorecan be used for highly water-insoluble drugs. The choice of vehicle isoften dose limiting for drugs that have little inherent toxicity, sincevehicle toxicity can limit the maximum deliverable dose of drug.

To assess toxicity, a small drug-scaling analysis in intact, normal (notimmune-compromised) female NCr mice (our standard strain) is performed.The primary goal is to establish a maximum tolerated dose (MTD) belowwhich all subsequent activity studies will be performed. A change in therate of weight gain is used as the initial measure of toxicity. Animalsare monitored for behavioral or other changes that can indicatetoxicity, e.g.,somnolescence, limited mobility, hunching, and generalchange in appearance consistent with poor health. Some types oftoxicity, however, may be transient and resolve within a few hours.Doses are increased at 10-fold increments until evidence of toxicity(toxic dose) is observed or the maximum deliverable dose is achieved.The highest dose that yields no detectable toxicity is the initial MTDestimate.

Blood is collected from treated animals in heparinized tubes,centrifuged, and the serum stored at −80° C. All tissues are removed atnecropsy, visually examined, and placed in formalin. Subsequently,formalin-fixed tissues can be embedded in paraffin, sectioned, stainedwith H & E, and examined by a pathologist. Serum is used for standardblood chemistry or to measure several relevant end-points that might beaffected by ER-α modulators, e.g., serum HDL- and LDL-cholesterol andtriglycerides.

Doses at which body weight change or other physiologic/behavioraltoxicity occurs are compared with those doses at which blood chemistrychanges or gross or microscopic anatomic changes in tissues occur. TheMTD may be adjusted downward if changes are detected in these end-pointsat doses lower than those utilizing the less sensitivephysiologic/behavioral end-points. These studies may also provide someinsight into the potential mechanisms of toxicity.

Example 8

In vivo activity studies will be performed in 4-6 week old female(ovariectomized) NCr athymic nude (nu/nu) mice. Animals receive mammaryfat pad inocula (orthotopic) of tumor cell suspensions, e.g., MCF-7cells since these cells are E2-dependent and allow for the directtesting of the test compounds on ER-α regulation of cell survival invivo. Mice are given a single inoculation of 5×10⁶ tumor cells in≤100-μl of cell culture growth medium. The mice are implantedsubcutaneously with a 17β-estradiol (0.72 mg)/60-day release pellet(Innovative Research of America) 48-hr before inoculation of tumorcells, to allow MCF-7 cell growth.

Example 9

The end-points include specific tumor growth delay, tumor incidence, andtumor doubling time. Tumor area is recorded every 2-3 days by measuringthe longest axis and perpendicular width. Tumor doubling time (Td) isestimated following Gompertzian transformation. At the end of eachexperiment, the organs and tumors are removed for histopathologicalexamination. For specific tumor growth delay in vivo, the times takenfor both treated (T) and control (C) tumors to reach a predeterminedsize are measured. The growth delay is measured as (T-C)/C where the T/Cor T-C values are median values. Repeated measures ANOVA are used tocompare tumor size at each time point across the analysis and/or tumordoubling times are estimated and compared. Td values are compared byeither one-way or multivariate ANOVA. Tumor latency and specific growthdelay are explored using the Kaplan-Meier approach, and differencesamong groups tested by the Log-Rank test. Tumor incidence data(proportion of proliferating tumors/group) are compared using aChi-squared test.

For tumor growth delay studies, drug treatment begins when the tumorsreach 0.5 cm in diameter. The dose is set at half of the MTD, and thefrequency of dosing is determined empirically in pilot studies, using upto daily doses for a maximum of 60 days.

What is claimed is:
 1. A method of treating a condition marked byabnormal cell growth, the method comprising administering to the subjecta composition comprising a therapeutically effective amount of thecompound

or a pharmaceutically acceptable salt thereof.
 2. The method of clam 1,wherein the administered compound is a hydrochloric acid (HCl) salt. 3.The method of claim 1, wherein the cells that are growing abnormally areestrogen-receptor positive (ER+) cells.
 4. The method of claim 1,wherein the genome of the cells contains a BRCA1 mutation.
 5. The methodof claim 1, wherein the cells are resistant to tamoxifen or fulvestrant.6. The method of claim 1, wherein the compound is co-administered withan agent selected from the group consisting of a vinca alkaloid, anucleic acid inhibitor, a platinum agent, interleukin-2, an interferon,an alkylating agent, an antimetabolite, a corticosteroid, a DNAintercalating agent, an anthracycline and a urea.
 7. The method of claim1, wherein the compound is administered intravenously, intramuscularly,intraperitonealy or orally.